DNA fragmentation and multifaceted toxicity induced by high-dose vanadium exposure determined by the bioindicator Allium test

In this study, the toxicity of vanadium (VCI3) in Allium cepa L. was studied. Germination-related parameters, mitotic index (MI), catalase (CAT) activity, chromosomal abnormalities (CAs), malondialdehyde (MDA) level, micronucleus (MN) frequency and superoxide dismutase (SOD) activity were investigated. The effects of VCI3 exposure on the DNA of meristem cells were investigated with the help of comet assay, and the relationships between physiological, cytogenetic and biochemical parameters were revealed by correlation and PCA analyses. A. cepa bulbs were germinated with different concentrations of VCI3 for 72 h. As a result, the maximum germination (100%), root elongation (10.4 cm) and weight gain (6.85 g) were determined in the control. VCI3 treatment caused significant decreases in all tested germination-related parameters compared to the control. The highest percentage of MI (8.62%) was also observed in the control. No CAs were found in the control, except for a few sticky chromosomes and unequal distribution of chromatin (p > 0.05). VCI3 treatment caused significant decreases in MI and increases in the frequencies of CAs and MN, depending on the dose. Similarly, the comet assay showed that DNA damage scores increased with increasing VCI3 doses. The lowest root MDA (6.50 µM/g) level and SOD (36.7 U/mg) and CAT (0.82 OD240nmmin/g) activities were also measured in the control. VCI3 treatment caused significant increases in root MDA levels and antioxidant enzyme activities. Besides, VCI3 treatment induced anatomical damages such as flattened cell nucleus, epidermis cell damage, binuclear cell, thickening in the cortex cell wall, giant cell nucleus, damages in cortex cell and unclear vascular tissue. All examined parameters showed significant negative or positive correlations with each other. PCA analysis confirmed the relations of investigated parameters and VCI3 exposure.


Material and methods
Test material. A. cepa bulbs (2n = 16), which is a eukaryotic indicator organism, were preferred as test material. Bulbs were obtained from a local market in Giresun.
Test chemical. Vanadium (III) chloride (VCI 3 -CAS No: 7718-98-1-25 G), a product of Merck company, was used as test chemical. VCI 3 doses were determined according to EC 50 . The EC 50 concentration for VCI 3 was calculated as 100 µg/L. Along with the EC 50 dose, a dose of 200 µg/L, which is twice the EC 50 , and 50 µg/L, which is half of it, were preferred.
Experimental process. In the study, healthy and equal-sized bulbs were used and their outer shells were removed before the application. The bulbs were divided into 4 groups and control bulbs were treated with tap water. Vanadium application was used at concentrations determined according to the EC 50 value and for this purpose, the bulbs in the application group were germinated with VCI 3 at doses of 50 µg/L, 100 µg/L and 200 µg/L. The bulbs of the control and the treatment groups were germinated at 24 °C for 72 h. At the end of the period, the bulbs and root tips collected from each group were used for experimental analysis, measurement and observations 19 . Experimental research on plant samples, including the supply of plant material, complies with institutional, national and international guidelines and legislation. In this study, the toxic effects and possible mechanisms of VCI 3 were elucidated by using different parameters.
Physiological parameter measurements. Root growth was assessed by determining the radicle length and the weight gain was determined by measuring the weights of each bulb before and after the application. The germination percentage (GP) was calculated using Eq. (1) 19,20 . In the germination percentage test 50 bulbs; in root length and weight gain analysis 10 bulbs were tested.
Cytogenetic parameter observations and counts. Mitotic slides were prepared from root tip samples to observe CAs and MN and to examine MI rates. After germination, root tip samples of each group were collected and subjected to routine fixation, hydrolysis and staining processes. Acetocarmine was used for staining www.nature.com/scientificreports/ mitotic chromosomes in root tip cells. Mitotic slides were examined under a research microscope 19,21 . Observations and counts of CAs and MN were performed by two different observers to ensure their accuracy. The criteria determined by Fenech et al. 22 were taken into account in the determination of MN formations. 1000 cells were analyzed for MN and CAs analysis. MI was calculated with Eq. (2) and calculated by analyzing 10,000 cells in each group.
Comet test. Comet analysis was applied according to the protocol suggested by Chakraborty et al. 23 . Root tip samples were crushed gently in 400 mM Tris-buffer for the isolation of nuclei to obtain nuclear suspension. Slides immersed in 1% NMPA solution were dried at room temperature overnight. 40 µL of nuclear suspension and 40 µL of 1% LMPA from each group were gently mixed and added to each slide. A coverslip was placed over the mixture to obtain a uniform layer. After solidification, the coverslip was removed and a layer of 80 µL 0.5% LMPA was formed on the surface. Slides containing the nuclear suspension were transferred to gel electrophoresis tank with Na 2 EDTA and NaOH (pH > 13 Biochemical parameter measurements. MDA levels were measured according to the procedure recommended by Unyayar et al. 26 . 0.5 g of the sample was homogenized with 5% trichloroacetic acid. 0.5% thiobarbituric acid was added to the supernatant obtained after centrifugation and then incubated at 96 °C in 20% trichloroacetic acid. The absorbance of the supernatant obtained by centrifugation at the end of the incubation was measured at 532 nm 19 . The root tips of each group were collected and extracted for enzyme activities. For this purpose, 0.5 g of sample was homogenized and the supernatant was used for biochemical analysis 19,27 . SOD enzyme activities (U/mg FW) were measured according to the procedure proposed by Beauchamp and Fridovich 28 . CAT activity (OD 240nm min/g FW) was measured according to the method developed by Beers and Sizer 29 . All analyzes were performed in triplicate to determine the levels of SOD, CAT and MDA.
Anatomical examinations. Cross-sections of root samples from each group were taken to investigate the effects of vanadium exposure on root anatomy. Root samples were washed three times consecutively with distilled water to remove surface residues then cross-sections were taken from root tips collected from bulbs belonging to each group. Sections placed between the slide and the coverslip were stained with 5% methylene blue and made into a fixed preparation with Canadian balm. Anatomical structures were examined with the IM-450 TI model research microscope 30 .
Statistical analyzes. SPSS Statistics 22 (IBM SPSS, Turkey) package program was used for statistical analysis. Statistical significance between data as mean ± standard deviation (SD) was determined by one-way analysis of variance, "One-way Anova" and "Duncan" tests. P values less than 0.05 were considered statistically significant. Correlation and principal component analyzes (PCA) were performed with Rstudio v 1.4.1106 program 31 . Pearson correlation analysis (bilateral) and graphs were obtained with hmisc and corrplot packages 32 . PCA was performed according to physiological, biochemical and genetic parameters, which are different toxicity biomarkers tested. PCA analysis was performed using the factoMineR 33 and factoextra 34 packages available in RStudio.

Results and discussion
Physiological findings. The effects of VCI 3 application on germination-related parameters are shown in Table 1. A normal germination process and the highest germination parameters were obtained in the control. Physiological parameters tested in this study regressed in the groups treated with VCI 3 . The most significant  www.nature.com/scientificreports/ regression was detected in the group treated with 200 µg/L VCI 3 . In Group IV, germination rate, root length and weight gain decreased by 28%, 2.6 times and 2.3 times, respectively compared to control. Our results are similar to literature studies investigating the physiological effects of vanadium and vanadium compounds. Vachirapatama et al. 35 reported a decrease in stem length, leaf number, leaf, stem and root dry weight as the vanadium concentration increased in green mustard and tomato plants exposed to ammonium meta vanadate (NH 4 VO 3 ). Macar et al. 14 reported that 25, 50 and 100 µg/L vanadyl sulfate pentahydrate (VOSO 4 · 5H 2 O) exposure caused significant reductions in germination-related parameters in A. cepa bulbs. Aihemaiti et al. 36 reported a 50% reduction in root length of Setaria viridis grown in soil containing 40-55.8 mg/L vanadium. Abnormalities observed in germination parameters as a result of VCI 3 exposure may be associated with the decrease in nutrient intake of root, damages in the root structure and regression in mitotic cell division. The biotransformation process including reduction reactions takes place in the uptake of vanadium by plant roots, and aldehyde, ketone, catechol, olefin and sulphide groups are oxidized in these regions. These reactions provide a strong attachment and immobilization of vanadium to root tissues. In this way, vanadium remaining in the root for a long time causes serious toxic effects 5 . Delays and abnormalities in root growth also reported in literature studies confirm this claim. Vachirapatama et al. 35 determined that high doses of vanadium damaged the structure of tomato roots and prevented them from working efficiently. In our study, structural abnormalities such as epidermis and cortex cell damage were observed in A. cepa. The same researchers determined that vanadium inhibits plasma membrane ATPase activity, which is known to have an important role in the uptake of nutrients by plant cells, and fructose 2,6-biphosphatase, which is involved in photosynthesis. Also heavy metal ions inhibit root growth by slowing root meristem cell division 19,37 . In our study, a decrease in MI values of root meristem cells was observed due to the increase in VCI 3 dose. Table 2. The highest MI, lowest MN frequency and CAs levels were obtained in the control. VCI 3 exposure caused a decrease in MI and an increase in MN and CAs numbers (p < 0.05). Vanadium administration caused serious reductions in MI rates, confirming the regressions in physiological parameters (Fig. 1). Among the VCI 3 application groups, the highest decrease in MI value and the highest increase in the frequency of MN and the number of CAs were determined in 200 µg/L VCI 3 -treated group. In Group IV, MI decreased by 2.3%, while the frequency of MN increased 31 times and fragment damage, the most observed CAs, increased 29 times compared to the control. The stages of mitosis in the control are given in Fig. 2   www.nature.com/scientificreports/ application are given in Fig. 3. Among CAs abnormalities, a high rate of fragments was observed, while reverse polarization was observed at the lowest level.

and the different types of CAs formations induced by vanadium
Our results are compatible with the findings of literature studies investigating the genotoxicity induced by vanadium and vanadium compounds. Marcano et al. 38 did not detect any change in MI values of A. cepa exposed to vanadium solutions, while they found an increase in the frequencies of CAs. Roychoudhury 5 reported that meristematic cells of Lens culinaris (lentil) seeds exposed to vanadium stress had various CAs such as break, bridge, sticky chromosome and ring chromosome. On the other hand, studies of vanadium-induced genotoxicity have focused more on cultured human peripheral lymphocyte cells. Rodriguez-Mercado et al. 39 reported that vanadium (IV) tetraoxide (V 2 O 4 ) induced a decrease in mitotic levels of human lymphocyte and leukocyte cells in culture and a significant increase in the frequency of CAs. In another study by Rodríguez-Mercado et al. 40 , structural CAs such as break, acentric fragment, dicentric and gap, and a decrease in MI were detected in human peripheral lymphocyte cells exposed to different compounds of vanadium.
The alteration in MI value and the induction of MN and CAs formations by VCI 3 treatment can be explained by the damage in DNA and microtubules (α-tubulin and β-tubulin proteins) due to induced oxidative stress. Because Çanlı 41 reported that high vanadium concentrations significantly inhibited cell division. Similarly, Rodríguez-Mercado et al. 38 associated the decrease in MI as a result of vanadium exposure with the decrease in cell proliferation rate. Imtiyaz et al. 13 determined that vanadium stress causes DNA fragmentation by changing DNA integrity. Explaining the possible mechanisms of vanadium genotoxicity, Rodríguez-Mercado et al. 40 reported that the vanadium ion can bind to DNA by interacting with the N-7 atoms of guanine and adenine bases and the PO 2 groups in the backbone, or to form hydroxyl radicals (·OH) by reacting with hydrogen peroxide. They also stated that the vanadium ion produces ·OH radicals, especially through fenton-type reactions, and these radicals can cause single and double-chain breaks in the DNA skeleton, sugars and bases by damaging the phosphate groups in DNA.    44 reported that vanadium exposure caused double chain breaks of splenocyte cells of mice, resulting in an increase in comet tail lengths. Rivas-Garcia et al. 45 observed that exposure to vanadium in their comet test analysis caused an increase in DNA tail moment by promoting DNA damage in the HepG2 cell line. While MDA, an indicator of lipid peroxidation, was 6.5 µM/g in the control, VCI 3 administration increased the MDA level significantly. The most significant increase was observed in the 200 µg/L VCI 3 -treated group, and the MDA level in this group increased by 65.4% compared to the control and was measured as 18.8 µM/g. Lipid peroxidation is an indicator of oxidative stress, and antioxidant enzyme activities were also induced against increased oxidative stress. In Group IV, when compared with the control, SOD activity increased 1.9 times and CAT activity increased approximately 2.7 times. Similarly, Abedini et al. 46 reported that MDA levels increased in sunflower plant exposed to ammonium metavanadate (NH 4 VO 3 ) at 3.25, 7.5 and 15 mg/L doses, and also antioxidant enzyme activities enhanced. Imtiaz et al. 47 observed that SOD and CAT activities increased linearly with increasing NH 4 VO 3 concentrations in Cicer arietinum L. (chickpea) when grown in vanadium-contaminated soil. Altaf et al. 48 observed that NH 4 VO 3 exposure for two weeks caused an increase in enzyme activities and MDA levels of pepper seedlings at all dose levels.

Biochemical findings. The effects of VCI 3 treatment on antioxidant-oxidant parameters are shown in
MDA is a three-carbon dialdehyde and formed by the peroxidation of lipids in the cell membrane. In other words, MDA is a reliable indicator for assessing cell damage due to lipid peroxidation 49 . Studies have shown that MDA can alter DNA and RNA, cause cross-links in proteins and lipids, and suppress many genes involved in the plant's response to stressors. It is also known that MDA is carcinogenic and mutagenic 50 . One of the most important effects of metal toxicity is the production of ROS and its ability to induce irreversible damage to macromolecules such as DNA and protein in plants. Plants resort to antioxidant defense system enzymes to scavenge the produced ROS. Two of the most important of these enzymes are SOD and CAT 51 . In this study, the increase in MDA levels and SOD and CAT enzyme activity as a result of VCI 3 exposure may be due to the increased free radical levels in the cell. H 2 O 2 is the main component of these free radicals and is produced in excess when plants are exposed to heavy metals. Produced these free radicals can also cause membrane damage in the roots and increase lipid destruction, which leads to MDA production 46,47 . In order to defend itself against these damages of free radicals, the plant activates antioxidant enzyme genes and increases antioxidant enzyme synthesis. The Anatomical findings. The damages induced by VCI 3 exposure and the severities of these damages are given in Table 3. VCI 3 exposure caused different types of anatomical damages (Fig. 6) and the severity of the damages increased in a dose-dependent manner. The frequency of anatomical damages varies in each group. While giant nucleus, unclear vascular tissue and binuclear cells were not observed in the 50 µg/L VCI 3 -treated group, flattened cell nucleus, epidermis and cortex cell damages and thickening of the cortex cell wall were detected  www.nature.com/scientificreports/ at a small level. In the group in which 200 µg/L VCl 3 was applied, unclear vascular tissue and binuclear cells were detected at a moderate level, while other abnormalities were detected at severe level. In the literature, the anatomical changes induced by vanadium compounds in the roots of different plant species have been discussed at the macroscopic or morphological level rather than the cellular level. Therefore, the number of studies at the cellular level is extremely small. In one of these studies carried out by our study team, Macar et al. 14 have reported that exposure to vanadium sulfate pentahydrate at 25, 50 and 100 µg/L doses caused cellular damages such as epidermis cell damage and enlargement of the cell nucleus volume in meristem cells. In another study, Li et al. 52 observed that vanadium dioxide nanoparticles damaged the root structure by causing shrinkage in xylem conduction tissue cells in pea seedlings. In the study performed at the macroscopic level, Vachirapatama et al. 35 found that exposure to NH 4 VO 3 in the 20-80 mg/L dose range caused the darkening of tomato roots and disrupted the root structure. The biotransformation process including reduction reactions takes place in the uptake of vanadium by plant roots, and these reactions provide strong attachment and immobilization of vanadium to root tissues. In this way, vanadium remaining in the root for a long time causes serious anatomical effects 5 . As a result of VCI 3 treatment, the cell deformation, flattened nucleus, giant nucleus and binuclear cell abnormalities observed in root meristem cells show that vanadium remains in the cell and causes abnormalities, especially in the nucleus. The increase in MDA level induced by vanadium indicates an increase in lipid peroxidation and oxidative stress, and DNA fragmentation indicates deterioration in DNA integrity. These damages make the changes in the dynamics of nucleus inevitable and shape changes may occur in the nucleus, which is spherical or ellipsoidal under normal conditions 19,20 . A significant increase in the number of epidermis cells was determined in the groups exposed to VCI 3 and these cellular increases act as a barrier that reduces the entry of vanadium into the cell. These increases also increase the contact of cells with each other and deformations may occur in the nuclei of the cells and these cells due to formed mechanical pressure. Thickening of the cortex cell wall associated with the accumulation of structures such as cellulose and suberin to provide support and strength to the cells is a defense mechanism that the plant develops directly to reduce vanadium entry 53 . While unclear vascular tissue was not observed in the 50 µg/L VCI 3 -treated group, it was observed at a low level in the 100 µg/L VCI 3 and moderate level in the 200 µg/L VCI 3 -treated group. With the mechanisms developed by the plant such as thickening of the cortex cell wall and tightening of the epidermis cells the vanadium was prevented from reaching the conductive tissue, therefore, no vascular tissue damage was observed at a dose of 50 µg/L. However, with the increase in dose, the tolerance mechanism was insufficient and damages were observed in the vascular tissue in 100 µg/L and 200 µg/L VCI 3 www.nature.com/scientificreports/ applications. This uncertainty in the vascular tissue can be explained by the fragmentation of cell membranes, accumulation of materials or necrotic regions 54,55 .

Correlation and principal component analysis of parameters.
Correlation analysis of the investigated physiological, cytogenetic and biochemical parameters is shown in Fig. 7a. Positive correlations are expressed in blue, while negative correlations are represented in red. Color intensity and circle size are given in proportion to the correlation coefficients. When the correlation data were examined, it was determined that all parameters had significant positive or negative effects on each other. VCI 3 exposure was negatively correlated with weight gain, root length and MI, and positively correlated with DNA damage, MN frequency, CAs number, MDA, SOD and CAT levels. In addition, weight gain, root length, MI index parameters, among the parameters examined in the study, showed negative correlations with MN frequency, DNA damage score, CAs number, VCI 3 dose, and MDA, SOD and CAT levels. In the current study, the statistical data reduction method PCA was used to reduce the complexity of data interpretation in multiple biomarkers analyzes and is given in Fig. 4b. PCA analysis provided a more reliable and complete understanding of the toxicity status and the relationships between biomarkers. Relationships between applied VCI 3 doses and two physiological, three biochemical and four genetic parameters were investigated using PCA analysis. In Fig. 7b

Conclusion
The excessive use of petroleum products, especially in industry and transportation, pesticide and fertilizer applications containing vanadium in agriculture cause excessive accumulation of vanadium in the air, water and especially in the soil. This contamination can reach all living things through the food chain and cause various toxic effects. In this study, it was determined that vanadium chloride caused a wide-ranging toxic effect on A. cepa, a bio-indicator organism. Vanadium exposure caused a decline in physiological parameters related to plant growth. One possible reason for this regression is the decrease in the mitotic index, and there were significant reductions in dividing cell numbers in the vanadium-treated groups compared to the control. These reductions may be associated with the formation of CAs and MN induced by vanadium. Such genome instabilities cause cell cycle delays and a direct decrease in cell proliferation. Vanadium exposure also caused deterioration in the antioxidant/oxidant balance in stem cells, and the increase in MDA level and the increase in antioxidant enzyme activities indicate oxidative stress. The main reason for the physiological regressions and cyto-genotoxic effects is the oxidative stress induced by vanadium. The cell has developed mechanisms to cope with oxidative stress and to develop tolerance to vanadium, such as induction of antioxidant enzymes and alteration of various anatomical structures. Since this study is the most comprehensive study that deals with the toxicity of VCI 3 in plants with www.nature.com/scientificreports/ the help of many different parameters and provides information about the possible mechanism of toxicity, it provides important contributions to the literature and serves as a guide for future studies.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.